The survival and functioning of cells relies on the elaborate intercellular and intracellular communication network known as signal transduction pathways. These pathways coordinate proliferation, differentiation, metabolism, cell adhesion and motility, protein sorting and transportation of multitudes of cells in different tissues and organs. The malfunctions of these pathways can result in many diseases such as cancer and rheumatoid arthritis. Therefore, great effort has been focused on the development of therapies based on blocking cellular signaling in the diseased cells. Levitzki, A., Targeting signal transduction for disease therapy, Current Opinion in Cell Biology, 1996, 8, 239-244.
Intracellular signaling is a series of coupled events that transduce stimuli from outside of the cell into the cell. The signals are relayed by different sets of signal transducer proteins in a sequential fashion, and result in cellular responses. In this process, post-translational modifications of proteins by addition and removal of phosphate groups provide molecular switches to turn on and turn off the function of various transducer proteins. A broad family of protein phosphatases catalyzes the removal of phosphate groups from serine, threonine or tyrosine residues on proteins.
CDC25 comprises a family of dual specificity protein phosphatases which act to remove inhibitory phosphates from cdk kinases and activate cyclin dependent kinase complexes that trigger cell cycle progression. Sadhu, K. et al., Human homolog of fission yeast cdc25 mitotic inducer is predominantly expressed in G2, Proc. Nat. Acad. Sci. U.S.A. 1990, 87, 5139-5143; Galaktionov, K. and Beach, D., Specific activation of cdc25 tyrosine phosphatases by B-type cyclins: evidence for multiple roles of mitotic cyclins, Cell 1991, 67, 1181-1194. In human cells, CDC25 consists of three phase specific isoforms, CDC25A, B and C. CDC25A is expressed early in the G.sub.1 phase of the cell cycle, and is essential for transition from G.sub.1 to S phase. Jinno, S. et al., Cdc25A is a novel phosphatase functioning early in the cell cycle, EMBO J, 1994, 13, 1549-1556.
The activation of at least two cyclin-dependent kinases, required for G1 to S phase progression, depends on the dephosphorylation of the catalytic cdk subunit at Tyr-15 and Thr-14 near the ATP-binding site. The phorsphorylation of these two residues blocks the binding of ATP to the cyclin-cdk complex and renders the complex catalytically inactive. The dephorsphorylation of both cyclin E-cdk2 and cyclin A-cdk2 complexes by CDC25A is tightly regulated in normal cells. Accumulating evidence suggests that inappropriate amplification or activation of CDC25A is characteristic of a number of cancers, including breast cancer. Galaktionov, K. et al, CDC25 phosphatases as potential human oncogenes, Science, 1995, 269, 1575-1577; Gasparotto, D. et al., Overexpression of CDC25A and CDC25B in head and neck cancers, Cancer Res, 1997, 57, 2366-2368; Galaktionov, K., et al., Cdc25 cell-cycle phosphatase as a target of c-myc, Nature, 1996, 382, 511-517; Hernandez, S. et al., cdc25 cell cycle-activating phosphatases and c-myc expression in human non-Hodgkin's lymphomas, Cancer Res 1998, 58(8):1762-1767. Thus increased CDC25A activities may contribute to the dysregulated growth of certain types of tumor cells. Both CDC25A and B phosphatases are considered promising target enzymes for the development of anti-cancer drugs. Draetta, G. and Eckstein, J. W., Cdc25 protein phosphatases in cell proliferation, Biochimica et Biophysica Acta, 1997, 1332, M53-M63.
Although CDC25 phosphatases have been identified as playing a crucial role in the replication cycle of cancer cells, studies of the intracellular function of CDC25 phosphatases have been severely hampered by the lack of potent inhibitors. To date few therapeutic agents have been reported as specific targets for these enzymes or as inhibitors of their activity. Vanadate, a broad-spectrum protein phosphatase inhibitor was the first CDC25A inhibitor found. Baratte B. et al., Screening for antimitotic compounds using the cdc25 tyrosine phosphatase, an activator of the mitosis-inducing p34cdc2/cyclin Bcdc13 protein kinase, Anticancer Res 1992, 12(3):873-880. In 1996, the natural product dysidiolide was reported as a cdc25A inhibitor with an IC.sub.50 value of 9.4 .mu.M. Gunasekera, S. P. et al., Dysidiolide: A Novel Protein Phosphatase inhibitor from the Caribbean Sponge Dysidea ehteria de Laubenfels, J.Am.Chem.Soc. 1996, 118, 8759-8760. Later, Rice and coworker also synthesized a group of inhibitors based on the pharmacophore of protein tyrosine phosphatase (PTPase) inhibitors. Rice, R. L. et al., A targeted Library of Small-Molecule, Tyrosine, and Dual-Specificity Phosphatase Inhibitors Derived from a Rational Core Design and Random Side Chain Variation, Biochemistry, 1997, 36, 15965-15974. This series of compounds inhibited CDC25A phosphatase competitively, the most potent inhibitor having an IC.sub.50 value of 15 .mu.M.
As discussed above, the dual specificity protein phosphatase (CDC25A) plays a crucial role in cancer cell proliferation, and there exists a need for efficient synthetic routes for more potent CDC25A inhibitors. However, the total synthesis of natural products is usually lengthy, and with lower yields. Readily available natural products, with defined stereochemistry and useful functional groups, can be utilized with novel and efficient chemical transformations to solve the difficult problems. The pyrolysis of alkyl azides to form cyano groups has been reported. Bock, H. and Dammel, R., Gas-Phase Pyrolysis of Alkyl Azide: Experimental Evidence for Chemical Activation, J.Am.Chem.Soc. 1988, 110, 5261-5269. However, no report has appeared on the pyrolysis of azido-steroids on the surface of silica gel.